Rapid recognition and signal transduction of mechanical wounding through various signaling molecules, including calcium (Ca²+), protein phosphorylation, and reactive oxygen species (ROS), are necessary early events leading to stress resistance in plants. Here we report that an Arabidopsis mitogen-activated protein kinase 8 (MPK8) connects protein phosphorylation, Ca²+, and ROS in the wound-signaling pathway. MPK8 is activated through mechanical wounding, and this activation requires direct binding of calmodulins (CaMs) in a Ca²+-dependent manner. MPK8 is also phosphorylated and activated by a MAPKK MKK3 in the prototypic kinase cascade, and full activation of MPK8 needs both CaMs and MKK3 in planta. The MPK8 pathway negatively regulates ROS accumulation through controlling expression of the Rboh D gene. These findings suggest that two major activation modes in eukaryotes, Ca²+/CaMs and the MAP kinase phosphorylation cascade, converge at MPK8 to monitor or maintain an essential part of ROS homeostasis.